Cattle-derived knob paratopes grafted onto peripheral loops of the IgG1 Fc region enable the generation of a novel symmetric bispecific antibody format.

In this work we present a novel symmetric bispecific antibody format based on engraftments of cattle-derived knob paratopes onto peripheral loops of the IgG1 Fc region. For this, knob architectures obtained from bovine ultralong CDR-H3 antibodies were inserted into the AB loop or EF loop of the CH3 domain, enabling the introduction of an artificial binding specificity into an IgG molecule. We demonstrate that inserted knob domains largely retain their binding affinities, resulting into bispecific antibody derivatives versatile for effector cell redirection. Essentially, generated bispecifics demonstrated adequate biophysical properties and were not compromised in their Fc mediated functionalities such as FcRn or FcγRIIIa binding.

A Single Amino Acid Substitution in Structural Protein VP2 Abrogates the Neurotropism of Enterovirus A-71 in Mice.

Enterovirus 71 (EV-A71) causes hand, foot, and mouth disease (HFMD) in children and has been associated with neurological complications. With no specific treatment and a monovalent vaccine limited to the Chinese market, HFMD remains a serious public health concern and an economic burden to affected societies. The molecular mechanisms underpinning EV-A71 neurovirulence have yet to be fully elucidated. In this work, we provide experimental evidence that a single amino acid substitution (I to K) at position 149 in structural protein VP2 of a non-mouse-adapted EV-A71 strain completely and specifically abrogated its infectivity in murine motor neuron-like NSC-34 cells. We showed that VP2 I149K mutant was impaired in murine SCARB2-mediated entry step but retained the ability to attach at the cell surface. In vivo, VP2 I149K mutant was fully attenuated in a symptomatic mouse model of progressive limb paralysis. While viral titers in limb muscles were comparable to mice infected with parental wild-type strain, significantly lower viral titers were measured in the spinal cord and brain, with minimal tissue damage, therefore indicating that VP2 I149K mutant is specifically impaired in its ability to invade the central nervous system (CNS). This study highlights the key role of amino acid at position 149 in VP2 in EV-A71 neurovirulence, and lends further support that the EF loop of VP2 represents a potential therapeutic target.

Identification of a Novel EF-Loop in the N-terminus of TRPM2 Channel Involved in Calcium Sensitivity.

As an oxidative stress sensor, transient receptor potential melastatin 2 (TRPM2) channel is involved in many physiological and pathological processes including warmth sensing, ischemia injury, inflammatory diseases and diabetes. Intracellular calcium is critical for TRPM2 channel activation and the IQ-like motif in the N-terminus has been shown to be important by mediating calmodulin binding. Sequence analysis predicted two potential EF-loops in the N-terminus of TRPM2. Site-directed mutagenesis combining with functional assay showed that substitution with alanine of several residues, most of which are conserved in the typical EF-loop, including D267, D278, D288, and E298 dramatically reduced TRPM2 channel currents. By further changing the charges or side chain length of these conserved residues, our results indicate that the negative charge of D267 and the side chain length of D278 are critical for calcium-induced TRPM2 channel activation. G272I mutation also dramatically reduced the channel currents, suggesting that this site is critical for calcium-induced TRPM2 channel activation. Furthermore, D267A mutant dramatically reduced the currents induced by calcium alone compared with that by ADPR, indicating that D267 residue in D267-D278 motif is the most important site for calcium sensitivity of TRPM2. In addition, inside-out recordings showed that mutations at D267, G272, D278, and E298 had no effect on single-channel conductance. Taken together, our data indicate that D267-D278 motif in the N-terminus as a novel EF-loop is critical for calcium-induced TRPM2 channel activation.

Protection against lethal enterovirus 71 challenge in mice by a recombinant vaccine candidate containing a broadly cross-neutralizing epitope within the VP2 EF loop.

Human enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) and is associated with several severe neurological complications in the Asia-Pacific region. Here, we evaluated that while passive transfer of neutralizing monoclonal antibody (nMAb) against the VP2 protein protect against lethal EV71 infection in BALB/c mice. Protective nMAb were mapped to residues 141-155 of VP2 by peptide ELISA. High-resolution structural analysis showed that the epitope is part of the VP2 EF loop, which is the "puff" region that forms the "southern rim" of the canyon. Moreover, a three-dimensional structural characterization for the puff region with prior neutralizing epitopes and receptor-binding sites that can serve to inform vaccine strategies. Interestingly, using hepatitis B virus core protein (HBc) as a carrier, we demonstrated that the cross-neutralizing EV71 antibodies were induced, and the VP2 epitope immunized mice serum also conferred 100% in vivo passive protection. The mechanism of in vivo protection conferred by VP2 nMAb is in part attributed to the in vitro neutralizing titer and ability to bind authentic viral particles. Importantly, the anti-VP2(aa141-155) antibodies could inhibit the binding of human serum to EV71 virions showed that the VP2 epitope is immunodominant. Collectively, our results suggest that a broad-spectrum vaccine strategy targeting the high-affinity epitope of VP2 EF loop may elicits effective immune responses against EV71 infection.